Introduction

The monocotyledonous plant sorghum (Sorghum bicolor L. Moench) belongs to the Poaceae family (Kvas et al. 2009). According to Meena et al. (2022), sorghum is a key ingredient in feed for poultry, cattle, and humans. It could also be a source of alternative bioenergy (Erickson et al. 2012; Zegada-Lizarazu and Monti 2012; Meena et al. 2022). The worldwide sorghum harvested area in 2021 was 40,925,310 ha, and the yield was about 1.49 t/ha according to FAOSTAT (2021). With 4330 kg/ha produced in 2021 and the harvested area of 2,626,440, the United States is one of the world’s top producers of sorghum (FAOSTAT 2021). In Hungary, the sowing area of sorghum is increasing year by year, from 9620 ha (in the year 2018) to 50,000 ha (in the last year) (Sorghum ID 2022; FAOSTAT 2022). Sorghum has been grown in Hungary since the end of the 19th century, but it has become a more important plant in recent years with the increase in the number of available varieties, as the old varieties had lower productivity and were late-ripening (Ivány et al. 1994; Hungarian Central Statistical Office 2022). In addition to its use as feed, sorghum is becoming more and more popular as a food due to reform and alternative eating habits such as a gluten-free diet. Because of its favorable dietary composition, the plant has great health benefits (Dahlberg et al. 2012; Astroteca et al. 2019; Visarda and Aruna 2019). Globally, the most significant yield of sorghum has been grown in the USA and Asia; on the other hand, Europe’s yield is about 1% (Visarda and Aruna 2019; FAOSTAT 2021).

The classic disease triangle, which is made up of the pathogen, the host, and the environment, is a striking example of how organisms are connected to their surroundings. According to Kissoudis et al. (2014), as the climate changes, biotic and abiotic stress factors will affect plants more often, and stress interactions will also happen more often. Sorghum panicles are sensitive to grain mold and head blight pathogens from anthesis to grain maturity (Frederiksen and Odvody 2000). In regions of Europe where farmers must deal with drought stress, an increase in temperature, and a decrease in precipitation, sorghum can be an excellent alternative crop (Rooney 2014). Sorghum is more resistant to heat and drought than C3 crops like wheat, which evolved in more temperate regions but are tropical in nature. In comparison to C3 crops, sorghum uses C4 photosynthetic systems that promote more effective CO2, solar radiation, water, and nitrogen utilization (Safian et al. 2022).

Most of the hybrids are developed for feed; in third-world countries, some hybrids are developed for food because, in some countries, such as India, sorghum covers most of the sowing area (Reddy et al. 2006; Visarda and Aruna 2019). Foodborne illness in humans and animals is still a health concern and can cause numerous problems in food- and feed-chain management. In the past few years, climate change and global warming have caused a large spread of different mycotoxin-producing species and the development of molds in sorghum grains during harvest time (Leslie et al. 2021). Moldy sorghum consumption has several serious consequences in both human and animal bodies (Prom et al. 2021).

Due to these facts, it is important to determine the plant pathogens in sorghum grains. Microscopic fungi can grow on sorghum grains that have been harvested, and the grains may even get damaged when they are stored. Aspergillus, Penicillium, and Fusarium species are examples of grain molds and pathogens that can cause head blight. These molds and pathogens reduce grain yield and quality and produce mycotoxins, which can make animals and people sick if they eat contaminated grain (Prom et al. 2021). In Europe there are some scientific publications about mycotoxin-producing fungi, such as Fusarium spp. (Ferrigo et al. 2023; Bottalico and Perrone 2002; Balaž et al. 1997). However, in the case of sorghum, little is known about the mycotoxin-producing fungi, according to Szécsi et al. (2010) F. verticillioides was identified from sorghum stalk.

The Fusarium genus is a large and complicated genus of plant pathogenic fungi found in nature. They are also cosmopolitan species, which is why they can appear everywhere. It also produces mycotoxins, which are becoming more and more dangerous to human health, and it can be allergenic as well (Desjardins 2006; Leslie and Summerell 2006).

Numerous species of the genus Fusarium are semi-saprophytic fungi that only become parasitic during a certain part of their life cycle. Species belonging to the genus are one of the largest and most influential groups of pathogens that infect most cereals, including sorghum (Tesso et al. 2010). When a dispositional (weak) condition occurs in the plant, it usually switches to a parasitic lifestyle. They are typical soil-borne pathogens, they are among the most common molds, and they are very important to the economy. Most of the time, they infect through wounds and can appear as plant pathogens as a result of secondary parasites (Munkvold 2003; Little and Magill 2009).

Sorghum cultivation may be associated with the ‘grain mold disease’ complex (GMDC), a globally known panicle disease complex in sorghum, and this complex has an acute disease called “Fusarium grain mold” (FGM). The essential Fusarium species responsible for the formation of the mycelial coating on the crown and grains are F. verticillioides, F. andiyazi, F. proliferatum, F. thapsinum, and F. sacchari; these pathogens can cause grain mold in sorghum (Summerell et al. 2011; Little et al. 2012; Chala et al. 2019; Pena et al. 2020; Corallo et al. 2023). F. verticillioides, the most prevalent Fusarium species that may be isolated from sorghum kernels, was identified by Italian researchers (Ferrigo et al. 2023).

In sorghum kernels, other mycotoxigenic species have been discovered in Brazil, Saudi Arabia, South Africa, India, Nigeria, Sudan, Kenya, Urugay, and Mexico, including Aspergillus flavus, Penicillium funiculosum, and Alternaria species (Astoreca et al. 2019). The isolated species in Europe that originated in Germany and Belgium are Aspergillus, Fusarium, and Alternaria species; in this instance, they were identified morphologically (Astoreca et al. 2019).

The objective of the present study is to determine the internal infestation of sorghum kernels caused by Fusarium, Alternaria and Aspergillus species and identify the some of the isolated Fusarium species. To reach this objective, infestation of sorghum kernels was studied; morphological identification of Aspergillus and Alternaria species and molecular species identification of six Fusarium isolates was characterized.

Materials and methods

Sample collection

The sorghum grains were collected from Martonvásár, Central Hungary (N 47° 18′ 50′′, E 18° 47′ 19′′), where fourteen sorghum plots (size 3 × 30 meters each) were next to each other in the variety experiment. The fourteen plots were sown with fourteen different varieties and hybrids. The soil is chalky chernozem with a humus content of 3.32% and a pH value of 7.1. The pre-crop was oat in this experiment. During the harvest the moisture ratio was 20%.

Sorghum grain samples were taken shortly before harvest, during which we collected the plants from five points on the plots (thus 14 × 5 samples) and milled them. Samples were stored in paper bags and transported to the Department of Integrated Plant Protection of the Hungarian University of Agriculture and Life Sciences (MATE) for further examination, where microbiological sampling was performed.

Pathogen isolation

Suface-sterilization of the kernels was performed with 10% NaOCl for 2 min after the kernels were rinsed twice with distilled water for 5 min. Individual surface-sterilized kernels were placed in PPA or Nash & Snyder’s medium (for a final volume of 1 liter of destilled water: 15 g peptone, 1 g KH2PO4, 0.5 MgSO4, 20 g agar, 50 ppm pentachloronitrobenzene (PCNB), and 100 ppm chloramphenicol) (Leslie and Summerell 2006) in 9 cm diameter plastic sterile Petri dishes. The experiment was performed on 10 grains per plate, and 10 biological replicates were used for each sorghum sample. The grains were placed in the Petri dish under sterile conditions, and then they were incubated at room temperature (25 °C), and 3 and 7 days later, the growing fungal colonies were examined. To identify the appearing fungi at the genus level, the fungal hyphae from the grains were placed on potato dextrose agar (PDA). In order to examine sporulation, the growing colonies were incubated for seven days at 25 °C with natural light supplemented with UV-A light. To identify the Aspergillus and Alternaria species on the genus level, morphological features of the colonies were examined under microscope (Olympus BX50) following the taxonomic keys for each genus (Samson et al. 1981).

DNA extraction from the fungal colonies

The extraction of DNA, the chloroform-isoamyl alcohol (CTAB) extraction protocol was performed based on Doyle and Doyle (1987). Total DNA was extracted from seven-day-old mycelium grown on a PDA.

PCR (Polymerase Chain Reaction) amplification

The PCR mixture (DreamTaq PCR Master Mix, manufactured by Thermo Scientific) contains all of the necessary reaction components and also contains the forward and reverse primers. The PCR was assembled according to the manufacturer’s instructions. A standard polymerase chain reaction (PCR) was used to amplify the TEF-1α (translation elongation factor) gene region for the identification of Fusarium species. During the experiment, the primers ef1 (forward primer; 5’-ATGGGTAAGGA(A/G)GACAAGAC-3’) and ef2 (reverse primer; 5’-GGA(G/A)GTACCAGT(G/C)ATCATGTT-3’) (O’Donnell et al. 2000) were used in a PCR reaction. The PCR was set on: 1 cycle of initial denaturation at 95 °C for 2 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 1 min, extension at 72 °C for 90 s, and 1 cycle of final extension at 72 °C for 5 min. During the PCR, ~700 bp of products were amplified.

To identify the Alternaria species the internal transcribed spacer (ITS) region was amplified using ITS5 (forward primer; 5’-CCGAGTGCGGCCTCTGGGTCC-3’) and ITS4 (reverse primer; 5’-TCCTCCGCTTATTGATATGC-3’) primers. The PCR was set on: 1 cycle of initial denaturation at 94 °C for 10 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 30s, extension at 72 °C for 1 min, and 1 cycle of final extension at 72 °C for 10 min. During the PCR, ~600 bp of products were amplified as previously described (White et al. 1990; O’Donnell et al. 1998).

A 1% agarose gel was prepared to detect amplified PCR products, and ethidium bromide (EtBr) (0.1 mg/ml) was used for staining. Evaluation of the gel was performed in a Bio-Rad gel documentation system using the Quantity One (Bio-Rad) program. The TEF and ITS PCR product was used as a template for DNA sequencing. Before sequencing the amplified samples were purified, the Sanger-dedoxy sequencing was made by Eurofins Biomi Ltd. Then, the sequences were compared and uploaded to the NCBI (US National Center for Biotechnology Information) database, which is accessible at https://blast.ncbi.nlm.nih.gov/Blast.cgi.

Statistical analysis

In the case of the sorghum samples a random sampling was used during our research. We used 10 grains in 10 repeats for each sample. We used RStudio to analyze the internal infections caused by the 3 major mycotoxin-producing fungi in Hungary. An analysis of variance (ANOVA) of the fungal isolation was presented with RStudio as well.

Results

Fungal infestation level, species spectrum and dominance in sorghum grain samples

Table 1 shows the internal infection values of sorghum grains. We have already isolated the fungi causing internal infection at the genus level. All the samples were 100% infected with Alternaria spp., Fusarium spp. and Aspergillus spp. During our experiments the Alternaria species dominated in the ratio of internal infections. However, Fusarium spp. internal infection was also found in all samples, though it was present in much smaller proportions in sorghum grains. Aspergillus spp. internal infection in sorghum grains was most rarely present in the examined samples and Aspergillus internal infection was detectable in most of the samples. It was statistically proven that all the samples have a significant difference (I: 95% confidence interval; SD: 1,079553).

Table 1 Means of the ratio of internal grain infections in percent caused by Fusarium spp., Alternaria spp., and Aspergillus spp. for 10 grains in 10 replicates. (I: 95% confidence interval; P < 0,05; SD: 1,079553)
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PCR assays

The isolated Fusarium strains were identified with molecular techniques. Table 2 indicates the identified species and their additional informations. From PCR amplification with the TEF-1α gene region from the 6 Fusarium isolates, a single band of 700 bp was successfully amplified (Fig. 1). The TEF-1α PCR product was used as a template for Sanger-dedoxy sequencing. Then, the sequences were compared to the NCBI (US National Center for Biotechnology Information) database. The BLAST search for similarities using NCBI BLAST indicated that the percentage of similarity of the isolates ranged from 97 to 100%. Based on the molecular identification and sequencing six Fusarium proliferatum strains were identified from sorghum grains during our study. According to the Alternaria spp. identification two isolates were amplified with the ITS region where a single band of 600 bp was successfully amplified. The ITS PCR products were used as a template for Sanger-dedoxy sequencing and the sequences were compared to the NCBI (US National Center for Biotechnology Information) database. The BLAST search for similarities using NCBI BLAST indicated that the percentage of similarity of the isolates ranged from 98%–100%. Based on the morphological examination and molecular identification of the isolates we identified Alternaria alternata species (Table 3).

Table 2 Identification of Fusarium isolates from sorghum based on morphological examination and TEF-1α sequences
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Fig. 1
figure 1

PCR products obtained with TEF-1α (translation elongation factor) region primer pairs (ef1/ef2) by 1% agarose gel electrophoresis from mycelial isolates (INVT_001, INVT_002, INVT_003, INVT_004, INVT_005 and INVT_006) with 2 positive and 2 negative controls at the end of the samples– with 1kb GeneRuler DNA ladder (Thermo Fisher Scientific). The expected amplification product is approximately 700 bp. The positive control samples are from the main collection of the Department of Integrated Plant Protection, Hungarian Unversity of Agriculture and Life Sciences, Hungary

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Table 3 Identification of Alternaria isolates from sorghum kernels based on morphological examination and ITS sequences
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Discussion

Despite the importance of sorghum-based products in food and feed, in Hungary little is known about mycotoxin-producing fungi isolated from sorghum kernels. Previous studies showed that the presence of Fusarium species isolated from sorghum kernels are common worldwide (Summerell et al. 2011; Little et al. 2012; Astoreca et al. 2019). This work represents one of the first study about the occurrence of mycotoxin-producing fungi in sorghum grain in Hungary. Nevertheless, this study is one of the first data on identification of Fusarium species in sorghum kernels in Hungary.

The presence of mycotoxin-producing fungi in sorghum kernels is a high risk due to the role of sorghum in food and feed chain. The present study were performed with TEF-1α (translation elongation factor) region primer pairs (ef1/ef2) to identify the present Fusarium species. Most of the studies have been done have focused on identification of Fusarium species in wheat and maize (Koncz et al. 2008; Szécsi et al. 2011). So far, no Fusarium species has been described or characterized from sorghum kernels in Hungary, only F. verticillioides strains were identified from sorghum stalks (Szécsi et al. 2010). Fusarium spp. were isolated from maize, rice, and wheat grains in Hungary, such as F. proliferatum, F. verticillioides, F. subglutinans, F. acuminatum, F. avenaceum, F. poae, and F. sporotrichioides (Koncz et al. 2008; Szécsi et al. 2011).

According to multiple literature, F. proliferatum has been consistently identified as the predominant Fusarium species found in sorghum grains (Leslie and Summerell 2006; Tesso et al. 2012; Kange et al. 2015). The aforementioned strain is observed in our collected samples, a finding that is corroborated by our experimental investigations. Based on the available literature, it has been shown that F. proliferatum, a fungal species identified in our experimental investigations, exhibits the ability to synthesize fumonisins, including fumonisin B1, a known carcinogen, as well as toxins B2, B3, B4, B5, and B6, which possess mutagenic properties. Certain farm animals, such as horses and pigs, exhibit a heightened susceptibility to mycotoxins. In humans, the presence of these toxins has been associated with the development of cancer (Bush et al. 2004). Fusarium species most commonly isolated from sorghum kernels is belonging to the Giberrella fujikuroi species complex and the Fusarium incarnatum-equiseti sepcies complex (Corallo et al. 2023; Funnell-Harris and és Pedersen 2011). Based on previous research, it has been documented that this particular fungal species has the the ability to produce multiple mycotoxins, including fumonisins. Additionally, some sources suggest that it is capable of producing beauvericin (BEA). Consequently, it is imperative to investigate the potential appearance of this fungal species in the food and feed-chain (Kvas et al. 2009; Fallahi et al. 2019). The significance of the beauvericin toxin’s characteristics requires consideration, particularly in light of its current lack of regulation by the European Union, in contrast to other mycotoxins such as ZEA and DON, as defined by EC 1881/2006.

The infestation experiments observed that Alternaria species predominated in the samples, they could produce several mycotoxins (Ostry 2008). According to the micro- and macromorphological and molecular identification with the ITS gene region, we identified two Alternaria alternata isolates from the examined sorghum kernels. Moreover, all the isolated Alternaria species were examined micro- and macromorphologically, from which we came to the conclusion that, compared to the molecularly identified isolates, these may also belong to the A. alternata species. The EFSA has already paid attention to the Alternaria alternata mycotoxins, which could be a link in the European Union’s (EU) food chain management. The “threshold of toxicological concern” (TTC) method was used by contaminants in the food chain to evaluate the relative level of risk for dietary exposure of humans to various mycotoxins.

Besides the infection of Alternaria and Fusarium species, we also observed internal infestations caused by Aspergillus species in sorghum kernels, which may pose additional food and feed safety risks. In 2012, an EFSA scientific report drew attention to the Aspergillus species and their toxigenic characteristics. The scenarios that were mentioned in the report are becoming reality nowadays (Battilani et al. 2012).

Investigating the presence of mycotoxin-producing fungi in sorghum is an important task, as mycotoxin contamination in the food and feed chains is one of the most important food and feed safety issues today. According to the EFSA, mycotoxins produced by Alternaria species (e.g., alternariol, alternariol monomethyl ether, and tenuazonic acid) are most common in cereals (EFSA et al. 2016). However, different regulations, such as those for mycotoxins produced by several Fusarium species, do not apply to mycotoxins produced by Alternaria species or Fusarium species, which can infect sorghum kernels.

Sorghum in Hungary is exposed to colonization by a wide variety of toxigenic genus such as Fusarium spp., Alternaria spp., and Aspergillus spp. that can cause the infestation of kernels and they could lead to grain contamination by several mycotoxins. These findings indicate to minimize the infection levels in sorghum grains in the field and the storage as well to avoid the mycotoxin contamination of sorghum kernels. This could ensure food and feed safety during sorghum cultivation.